The invention relates to compounds which are inhibitors of cell proliferation, having antiproliferative activity on a variety of cell types.
Many vertebrate cell types respond to diffusible growth factors as stimuli which regulate proliferation. A number of these growth factors and their cognate receptors have been purified, and the genes encoding them have been cloned and characterized (Sporn and Roberts eds. (1991) Peptide Growth Factors and their Receptors I and II. Springer-Verlaz, New York). Many cancers, which are diseases of cell proliferation, involve genetic modifications which affect the nature of the growth factor-receptor interaction. Such modifications can result in unregulated stimulation of proliferation in the receptor bearing target cell. Additionally, certain tumors of the nervous system involve the regulation of proliferation of cells from both the central and peripheral nervous systems.
The glial cells of vertebrates constitute the specialized connective tissue of the central and peripheral nervous systems. Important glial cells include the peripheral Schwann cells which provide both the metabolic support for neurons and the myelin sheathing around the axons of certain peripheral neurons, thereby forming individual nerve fibers. Schwann cells support neurons and provide a sheath effect by forming concentric layers of membrane around adjacent neuron axons, twisting as they develop around the axons. These myelin sheaths are a susceptible element of many nerve fibers. Damage to Schwann cells, or failure in growth and development, can be associated with significant demyelination or nerve degeneration characteristic of a number of peripheral nervous system diseases and disorders. In the development of the nervous system, it has become apparent that cells require various factors to regulate their division and growth. Several regulators of Schwann cell proliferation and differentiation have been identified. Such factors play an important role in both the development and the regeneration (following injury) of the peripheral nervous system.
Brockes et al. ((1984) J. Neuroscience 4:75-83) describe a protein growth factor present in extracts from bovine brain and pituitary tissue, termed Glial Growth Factor (GGF). This factor stimulates cultured rat Schwann cells to divide against a background medium containing ten percent fetal calf serum. GGF has been described as having a molecular weight of 31 KD and readily forming dimers. Brockes ((1987) Meth. Enz. 147:217-225) describes a Schwann cell-based assay for 31 kD GGF and purification using reversed phase HPLC.
The J. Neuroscience article of Brockes et al., supra, describes methods of purification of GGF to apparent homogeneity. In brief, one large-scale purification method described involves extraction of the lyophilized bovine anterior lobes and chromatography of material obtained thereby, using NaCl gradient elution from CM cellulose. Gel filtration is then carried out with an Ultrogel column, followed by elution from a phosphocellulose column, and finally, small-scale SDS gel electrophoresis. Alternatively, the CM-cellulose material was applied directly to a phosphocellulose column, fractions from the column were pooled and purified by preparative native gel electrophoresis, followed by a final SDS gel electrophoresis.
Brockes et al. ((1980) J. Biol. Chem. 255:8374-8377) observe that in gel filtration experiments the major peak of growth factor activity is observed to migrate with a molecular weight of 56 KD, whereas in the first of the above-described procedures activity was predominantly observed at molecular weight 31 KD. They report that the GGF dimer is largely removed as a result of the gradient elution from CM-cellulose in this procedure.
Benveniste et al. ((1985) PNAS 82:3930-3934) describe a T lymphocyte-derived glial growth promoting factor. This factor, under reducing conditions, exhibits a change in apparent molecular weight on SDS gels.
Kimura et al. ((1990) Nature 348:257-260) describe a factor they term Schwannoma-derived growth factor (SDGF) obtained from a sciatic nerve sheath tumor. The authors state that SDGF does not stimulate the incorporation of tritium-labelled TdR into cultured Schwann cells under conditions where, in contrast, partially purified pituitary fraction containing GGF is active. SDGF has an apparent molecular weight between 31 KD and 35 KD.
Davis et al. ((1990) J. Cell. Biol. 110:1353-1360) describe the screening of a number of candidate mitogens. The chosen candidate substances being examined for their ability to stimulate DNA synthesis in Rat Schwann cells in the presence of 10% FCS (fetal calf serum), with and without forskolin. One of the factors tested, GGF-carboxymethyl cellulose fraction (GGF-CM), was mitogenic in the presence of FCS, with and without forskolin. It was also observed that in the presence of forskolin platelet derived growth factor (PDGF) is a potent mitogen for Schwann cells. Previous to this finding, PDGF was not thought to have a mitogenic effect on Schwann cells.
Holmes et al. ((1992) Science 256:1205) and Wen et al. ((1992) Cell 69:559) demonstrate that DNA sequences which encode proteins which bind to a receptor (p185.sup.erbB2) are associated with several human tumors.
The p185.sup.erbB2 protein is a 185 kilodalton membrane spanning protein with tyrosine kinase activity. The protein is encoded by the erbB2 proto-oncogene (Yarden and Ullrich. (1988) Ann. Rev. Biochem. 57:443). The erbB2 gene, also referred to as HER-2 (in human cells) and neu (in rat cells), is closely related to the receptor for epidermal growth factor (EGF). Recent evidence indicates that proteins which interact with (and activate the kinase of) p185.sup.erbB2 induce proliferation in the cells bearing p185.sup.erbB2 (Holmes et al. (1992) Science 256:1205; Dobashi et al. (1991) Proc. Natl. Acad. Sci. 88:8582; and Lupu et al. (1992) Proc. Natl. Acad. Sci. 89:2287).
Although ligands have been identified which stimulate proliferation of cells with certain receptors (e.g., the p185.sup.erbB2 receptor), there exists a need to identify and isolate factors which act as inhibitors of cell proliferation at these receptor sites. Such inhibitors could be used for the purpose of treating cell proliferative disorders (e.g., neoplasms).